A role for microRNAs/Notch2R network in pediatric High-Grade Gliomas (pHGGs)

Gliomas represent a very heterogeneous group of tumors that affect the cellular components of the glial system. Pediatric High Grade Gliomas (pHGGs) are a rare, malignant and diffusively infiltrating neoplasms. They show an extremely broad range of clinical behaviour with only few patients achieving long-term survival. Notch signaling is an evolutionarily conserved pathway that regulates many cellular and developmental processes. Its dysregulation has been reported in many pathological contexts, including brain tumours, but little is known about the relevance of Notch signaling in pHGGs. The study of epigenetic mechanism engaged in its regulation could allow a better understanding of mechanisms at the basis of its deregulation. MicroRNAs are small molecules of 21-24 nucleotides in length and are emerging as major regulators of cancer-related gene expression. The identification of specific microRNAs signature is one of the latest hallmarks of scientific efforts. High-throughput microRNA profiling of pHGGs showing that several dysregulated microRNAs characterized these tumors. The present project intends to investigate the functional role of the Notch signalling unveiling epigenetic networks between dysregulated microRNAs and the Notch pathways. For the purpose, immunohistochemistry (IHC) on pHGG patients’ derived tissues and immunofluorescence (IF) on a patient derived pHGG cell line (KNS42) were performed to assess Notch receptors expression level. RT-qPCR was used to validate microRNAs levels in pHGG tissues and cells in respect to healthy brain tissues. Cell growth and viability were evaluated on KNS42 cells after microRNAs re-expression or pharmacological (GSI IX) and genetic (siRNA N2) treatments with by trypan blue exclusion assays. Luciferase reporter assay was performed to validate the binding of miR-29a-3p to the 3’-UTR of Notch2. IHC and IF analysis have demonstrated that Notch2 is highly expressed in pHGG tissues and cells. Pharmacologic and genetic treatments have highlighted its pivotal role in the control of pHGGs cell growth. Studies on patients’ derived pHGG tissues and pHGG cell line, KNS42, revealed down-regulation of three miRNAs, specifically miR-107, miR-181c and miR-29a-3p. Their re-expression in KNS42 cell line has effect on Notch2 protein levels and cell proliferation. The same effect was obtained after pharmacological inhibition of Notch signaling pathway with GSI IX for 96 h. Functional luciferase assay provides the evidence that miR-29a-3p is a direct negative regulator of Notch2 expression. The study reported suggests that Notch2 pathway activation plays a critical role in pHGGs growth and reveals a direct epigenetic mechanism that controls Notch2 expression. These findings identify new molecular targets for more effective treatment of these devastating pediatric brain tumors.